Identification of serum protein biomarkers by profiling N-glycoproteomes of patient-derived xenografts of clear cell renal cell carcinoma

PROJECT SUMMARY Postoperative surveillance (PS) by medical imaging is critical for the management of clear cell renal cell carcinoma (ccRCC) patients.

However, it is expensive and leads to increased risk for secondary malignancy due to accumulative exposure to radiation.

Similar challenges exists in monitoring treatment response for renal cell carcinoma (RCC) patients because the standard response evaluation criteria for solid tumors (RECIST) are based on changes of the anatomical tumor dimensions measured by routine CT or MRI imaging during treatment.

These imaging methods captured treatment effects insufficiently because of atypical response patterns during checkpoint inhibitor-based immunotherapy, the most effective therapy for RCC patients.

Compared to imaging, serum biomarker measurements eliminate radiation exposure to patients, are less expensive and most importantly, can reflect molecular changes due to therapies early on, sparing patients from ineffective treatments much sooner than using tumor volume reduction as an indicator.

N-glycoproteomic analyses of sera from patient-derived xenografts (PDXs) represent a new diagram of protein biomarker discovery that overcomes the most challenging limitation of proteomic profiling based approaches.

Our objective is to identify human-specific N-glycosylated proteins in a mouse background with high sensitivity using our ccRCC PDX models and validate potential biomarkers associated with tumor volume in ccRCC patient sera. PDX tumors will be generated from ten ccRCC PDX lines established in our laboratory using cryopreserved tissues.

Tumor tissue and serum will be collected when tumors reach particular volumes determined by MRI. N-glycoproteomes of PDX sera will be profiled using SAX-ERLIC based enrichment followed by LC-MS/MS.

Peptides identified will be assigned as human-specific, mouse-specific, or human- mouse conserved and subcellular localization of the human-specific proteins will be determined.

Similarly, N- glycoproteomes of PDX tumor tissues will be profiled and human-specific N-glycoproteins in ccRCC PDX tissues will be identified.

Proteins found in both PDX sera and tumor tissues but not in non-tumor bearing mouse sera will be compiles and target assays will be developed for the top candidate biomarkers.

The levels of these putative biomarkers in PDX sera and ccRCC patient sera will determined using targeted assays and their correlation with tumor volumes will be determined in PDX models and ccRCC patients. Our study represents the first attempt to use ccRCC PDX models to facilitate MS identification of serum biomarkers.

If successful, novel ccRCC-associated protein biomarkers will help reduce the cost of PS and treatment response assessment, decrease risk of developing second malignancies due to high cumulative exposure to ionizing radiation during medical imaging, allow early detection of molecular changes associated with recurrence or therapeutic treatment before clinical symptoms or change in bulk tumor volume, which in turn will greatly advance clinical management of ccRCC patients with the potential of prolonging survival.